The pre-placodal ectoderm (PPE) gives rise to the sensory organs in the vertebrate head, which frequently are subject to congenital defects and damage. In animal models, it has been demonstrated that the expression of the transcription factor Six1 is required for the establishment of the PPE and the formation of the sensory organs derived from it. Six1 transcriptional activity is modified by co-factors that bind to a highly conserved Six-domain located 5-prime to the DNA-binding homeodomain. In humans, two congenital defects characterized by craniofacial abnormalities and hearing loss (branchio-otic and branchio-otic-renal syndromes) are caused by mutations in SIX genes or one of the known cofactors, EYA1. However, these genes do not account for all of the cases of BO or BOR, indicating that other cofactors for SIX proteins may be causative. Recently, 25 potential binding partners of the Drosophila homologue of Six1, Sine oculis (SO), were identified by comprehensive yeast two-hybrid screens (Giot et al., 2003;Kenyon et al., 2005). Because of the high likelihood that the transcriptional activity of vertebrate Six1 is regulated in a very similar manner to fly SO, the investigator screened the Xenopus genomic database for the vertebrate homologues of the Drosophila SO binding partners that had the highest interaction confidence scores. The rationale was that this approach would quickly identify potential novel binding partners of vertebrate Six proteins that may be involved in establishing placode fate and subsequent differentiation. She evaluated the expression patterns of 14 Xenopus genes to determine which are expressed in the same domains as placodally expressed Six genes. Herein she propose to: 1) validate whether 12 of these candidate cofactors directly bind to Six1 or Six4;and 2) test by gain-of-function and loss-of-function whether these candidate cofactors have roles in placode formation and subsequent differentiation. This information will significantly advance our understanding of the normal development of cranial sensory structures, identify new genes that may be involved in human birth defects and hearing loss, and provide the preliminary data for an R01 application from a new investigator. PROJECT NARRATIVE: Placodal derivatives comprise important elements of the sensory specializations of the vertebrate head that are functionally necessary for an organism to react to and navigate its environment. It is particularly important to understand the molecular regulation of placode development because several genes involved in placode development are mutated in some human congenital syndromes. This application is designed to use information from two important animal models to identify new candidates for vertebrate craniofacial and cranial peripheral nervous system genetic defects.